Antibody fragments, compositions and uses thereof

ABSTRACT

Antibody fragment comprising a first polypeptide comprising a light chain variable domain and two constant domains and a second polypeptide comprising a heavy chain variable domain and two constant domains, wherein two chain constant domains are light chain constant domains and two constant domains are CHI heavy chain constant domains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/IB2014/058098, filed Jan. 7, 2014,which claims the benefit of Italian Patent Application No.TO2013A000012, filed Jan. 9, 2013.

FIELD OF THE INVENTION

The present disclosure concerns new antibody fragments with improved invivo stability.

BACKGROUND

Targeted therapy, the new frontier of cancer treatment, employspharmacological tools (drugs or antibodies) specifically blockingcrucial gene products that sustain the transformed phenotype. Currently,cancer targeted therapy is employed in the clinic for the treatment ofchronic myelogenous leukemias (CML), addicted to the tyrosine kinasemolecule ABL, for the treatment of a subset of Non-Small Cell LungCancers (NSCLC) and Colon-Rectum Carcinomas (CRC) relying on EpidermalGrowth Factor Receptor (EGFR/HER-1) activation and for the treatment ofBRAF-dependent melanomas.

Receptors with tyrosine kinase activity (RTKs) are interestingcandidates for targeted therapy as they are often hyper-activated inseveral types of tumors. They can be inhibited by different types oftargeting molecules, such as antibodies, that upon interaction with theextracellular part of the receptor are able to perturb thereceptor-induced intracellular signaling, and chemically-synthesizedsmall molecules that interfere with the receptor catalytic activity.

Among the different RTKs, the product of the c-met proto-oncogene, theHepatocyte Growth Factor Receptor (HGFR/Met), is emerging as one of themost important activated oncogene in cancer. Met controls a geneticprogram known as ‘invasive growth’ that includes pro-mitogenic,pro-invasive and anti-apotoptic cues. Through these physiologicalsignals, Met provides with a better fitness the tumor, helping it toovercome selective barriers in cancer progression. Moreover Met sustainstumor growth by its ability to promote tumor angiogenesis. In the lastyears, Met also resulted responsible for the aggressiveness developed bytumors treated with anti-angiogenic agents and for resistance toconventional radiotherapy. Additionally, MET gene alteration can be aprimary cause of transformation, in all of those cases in which it hasbeen genetically selected for the long term maintenance of the primarytransformed phenotype.

All the above listed findings have prompted the development of severalmolecules suitable to inhibit Met signaling, including competitiveinhibitors of HGF, chemical Met kinase inhibitors, anti-HGF and anti-Metantibodies. Some of these molecules, until now, have been tested onlyfor research purpose. Clinical trials are currently ongoing withneutralizing anti-HGF antibodies, anti-Met antibodies and several smallmolecules.

From several view-points, an anti-Met antibody able to inhibit Metsignaling would be preferable. Antibodies are highly specific, stableand, thank to their natural design, they are generally well tolerated bythe host. In the last years, several efforts have been put to generatetherapeutic anti-Met antibodies. However, a lot of failures have beenregistered, as the majority of the anti-Met antibodies behave asagonists, mimicking the HGF action. This is mostly due to the fact that,thanks to their bivalent structure, antibodies can stabilize receptordimers, allowing trans-phosphorylation of Met, with its consequentactivation. In one case, an agonist anti-Met antibody (5D5) has beenengineered and converted in a monovalent form (One Armed-5D5) that,competing with HGF binding, is endowed with therapeutic potential (1,2).This molecule has been recently entered a phase III clinical trial forthe treatment of a subset of Non Small Cell Lung Cancer patients,characterized by high level of Met expression in the tumors, incombination with erlotinib (3).

The monoclonal antibody DN30 is a mouse IgG2A directed against theextracellular moiety of the human Met receptor (4). It binds withsub-nanomolar affinity the fourth IPT domain of the Met receptorextracellular region. At the beginning, it was characterized as apartial agonist of Met, able to promote some, but not all, of theMet-mediated biological cell responses. Later it has been demonstratedthat it can act as an inhibitor of tumor growth and metastasis through amechanism of receptor ‘shedding’ (5). Receptor shedding is a physiologiccellular mechanism of protein degradation acting on diverse growthfactors, cytokines, receptors and adhesion molecules. Met shedding isarticulated in two steps: first a metalloprotease, the ADAM-10, cleavesthe extracellular domain of Met recognizing a specific sequencelocalized immediately upstream to the trans-membrane region; then theremaining transmembrane fragment becomes substrate of a second protease(γ-secretase) that detaches the kinase-containing portion from themembrane and rapidly addresses it towards the proteasome degradationpathway (6,7). The enhancement of this mechanism exerted by the DN30leads to a reduction in the number of Met receptors exposed at the cellsurface. At the same time, it releases a soluble, ‘decoy’ ecto-domain inthe extracellular space. The latter competes with the intacttrans-membrane receptor for ligand binding and inhibits receptorhomo-dimerization by forming hetero-dimeric complexes with bona fideMet. All these actions strongly impair Met-mediated signaling and resultin prevention of the downstream biological effects.

Recently the present inventors demonstrated that the monovalent Fabfragment of the DN30 anti-Met monoclonal antibody (DN30 Fab) is clearedof any agonistic activity and maintains the ability to induce shedding,thus resulting in a potent Met inhibitor (8). Induction of Met sheddingby DN30-Fab is dependent on the selective antibody-antigen interactionbut is independent from receptor activation. This mechanism of action,based on the simple elimination of Met from the cell surface, gives tothe DN30-Fab a strong advantage over other inhibitors, as it can beeffective against all the forms of Met activation, whether HGF-dependentor not, induced by overexpression, mutation or gene amplification.

While the recombinant DN30-Fab is very attractive for clinicalapplications, the short Fab plasma half-life—mostly due to renalclearance—severely limits its use for patient treatment.

Currently, the most consolidated technique to improve thepharmacological properties of a Fab fragment is to increase itsmolecular weight by conjugation with Poly Ethylen Glycol (PEG). FabPEGylation is a route pursued in most of the cases employing Fab in theclinic. The covalent attachment of the polymer chains to the antibodyfragment, obtained efficiently and without loss of antigen bindingproperties, is not an obvious process and requires a strong effort ofsetting up.

Another technique used to improve the pharmacological properties of aFab fragment is the one disclosed in EP-A-1 718 677. Such a procedure,used to generate the One Armed form of monoclonal antibody 5D5 commentedabove, is the production—on recombinant basis—of three differentantibody chains in the same cell, the light chain (VL-CL), the heavychain (VH-CH1-CH2-CH3) and the Fc portion of the heavy chain (CH2-CH3).The CH2-CH3 domains are not wild type: mutations, giving rise tospecific tridimensional structures, are included. In one polypeptide,the CH2-CH3 region incorporates a sequence forming a protuberance, whilein the other polypeptide the CH2-CH3 region contains a sequence forminga cavity, in which the protuberance can be inserted (Knob into holestructure). The presence of these tridimensional structures allows thepreferable formation of heterodimers in which the heavy chain formsdisulfide bonds with the Fc fragment, but does not exclude at all theformation of homodimers (i.e. two heavy chains linked together and twoFc linked together). Purification allowing the separation of theunwanted homodimers from the wanted heterodimers is mandatory. Thus the“One Armed procedure”, although very elegant, is cumbersome as itrequires additional steps in the overall process that complicate themanufacturing and reduce the yield of the recombinant antibody.

It is therefore felt the necessity of a different solution to increaseFab plasma half-life for in vivo therapeutic use.

SUMMARY OF THE INVENTION

The object of this disclosure is to provide an antibody fragment withimproved in vivo stability.

According to the invention, the above object is achieved thanks to thesubject matter recalled specifically in the ensuing claims, which areunderstood as forming an integral part of this disclosure.

An embodiment of the present disclosure provides an antibody fragmentcomprising a first polypeptide comprising a light chain variable domainand two constant domains and a second polypeptide comprising a heavychain variable domain and two constant domains, wherein two chainconstant domains are light chain constant domains and two constantdomains are heavy chain CH1 constant domains, fused in differentcombinations to the variable domains.

A further embodiment of the present disclosure concerns an antibodyfragment as defined above that is more stable in vivo than the Fabmolecule comprising the light and heavy chain variable domains.

A still further embodiment concerns an antibody fragment as definedabove that specifically binds the hepatocyte growth factor receptor(HGFR/Met).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in detail, purely by way of anillustrative and non-limiting example and, with reference to theaccompanying drawings, wherein:

FIGS. 1A-1B: Met shedding and down-regulation in Met-addicted cellstreated by chimeric MvDN30 or murine DN30 Fab. FIG. 1A SNU-5 a humangastric carcinoma cell line; FIG. 1B H1993-NC1 a non small cell lungcarcinoma cell line. Cells were incubated for 48 hrs in serum freemedium with the indicated concentrations of the two antibody fragmentsderived from DN30 mAb. Total Met levels were determined by Western blotanalysis of cell extracts using anti-Met antibodies. The two Met bandscorrespond to the unprocessed (p190 Met) and mature (p145 Met) forms ofthe receptor. Met shedding was determined by Western blot analysis ofconditioned medium using anti-Met antibodies. Both molecules efficientlyinduce Met-shedding/down-regulation.

FIGS. 2A-2B: Growth assay of Met-addicted cells treated by chimericMvDN30 or murine DN30 Fab. FIG. 2A EBC-1 a non small cell lung carcinomacell line; FIG. 2B Hs746T a human gastric carcinoma cell line. Cellswere plated in 96 well dishes (1000/well) in 10% FCS medium. After 24hrs cells were treated with increasing concentrations of antibodies forfurther 72 hrs. Number of cells was evaluated by Cell titer-glo (PerkinElmer). Each point is the mean of triplicate values; bars representstandard deviation. Both molecules efficiently inhibit cell growth ofMet-addicted cells.

FIG. 3: Schematic representation of the new DN30 derived molecules. Top:chimerized DN30 Fab (MvDN30); middle: Double Constant Domain Fab withthe duplicated constant domains in tandem (DCD-MvDN30.1); bottom: DoubleConstant Domain Fab with the duplicated constant domains swappedreciprocally (DCD-MvDN30.2). VH: variable domain of the DN30 heavychain. VL: variable domain of the DN30 light chain. CH1: constant domain1 derived from human IgG1 heavy chain. CL: constant domain derived fromhuman kappa light chain. Strep and His Tag: sequences included to allowprotein purification and immuno-detection.

FIG. 4: Analysis of the new DN-30 derived molecules. The indicatedpurified proteins were subjected to SDS-PAGE under reducing condition.Gel was stained with Gel Code blue (Pierce). All the molecules show twobands with the expected molecular weight.

FIG. 5: Binding to Met of DCD-MvDN30 molecules. ELISA binding analysisof MvDN30, DCD-MvDN30.1 and DCD-MvDN30.2 (liquid phase) to a Met-Fcchimera (solid phase). Binding was revealed using anti-strepTAGantibodies. O.D.: Optical Density; A.U.: arbitrary units. Each point isthe mean of triplicate values; bars represent standard deviation. Thenew molecules bind to Fc-Met with the same high affinity.

FIG. 6: Agonistic activity of DCD-MvDN30 molecules. A549 cells werestarved for 24 hrs and then stimulated for 10 min at 37° C. with thedifferent molecules at the indicated concentrations. Met activation wasdetermined by immuno-precipitation with anti-Met antibodies followed byWestern blotting with anti-Met antibodies specific for thephosphorylated Tyr 1234/1235 Met residues, the major phosphorylationsite (Top). The same blot was re-probed with anti-Met antibodies(Bottom). The new molecules do not significantly activate the Metreceptor.

FIG. 7: Agonistic activity of DCD-MvDN30 molecules. A549 cells werestarved for 24 hrs and then stimulated for 10 min at 37° C. with thedifferent molecules at the indicated concentrations. Activation of AKTand ERK-1,2 was determined by Western blotting with anti-AKT or anti-ERKantibodies specific for the phosphorylated form. The same blot wasre-probed with anti-Vinculin antibodies (Bottom) to control proteinloading. The new molecules do not significantly activate theMet-dependent signaling.

FIG. 8: Met shedding and down-regulation in cells treated by DCD-MvDN30molecules. A549 cells were incubated for 72 hrs in serum free mediumwith the indicated molecules (500 nM). Total Met levels were determinedby Western blot analysis of cell extracts using anti-Met antibodies. Thetwo Met bands correspond to the unprocessed (p190 Met) and mature (p145Met) forms of the receptor. As a loading control, the filter was probedwith an unrelated protein (actin). Met shedding was determined byWestern blot analysis of conditioned medium using anti-Met antibodies.The new molecules efficiently induce Met shedding.

FIG. 9: Inhibition of HGF-induced Met-activation by DCD-MvDN30molecules. A549 cells were incubated for 24 hrs in serum free mediumplus the indicated molecules (1000 nM) and then stimulated for 10 minwith HGF (100 ng/ml). Met activation was determined in total celllysates by Western blotting with anti-Met antibodies specific for thephosphorylated Tyr 1234/1235 Met residues, the major phosphorylationsite. The same blot was re-probed with anti-Met antibodies. Activationof AKT and ERK-1,2 was determined by Western blotting withanti-phosphoAKT or anti-phosphoERK antibodies. The same blot wasre-probed with anti-AKT or ERK-1,2 antibodies. To control proteinloading the filter was also probed with anti-Vinculin antibodies. Thenew molecules strongly inhibit HGF-induced Met-activation andMet-dependent signaling.

FIGS. 10A-10F: Anchorage-dependent growth of Met-addicted cells treatedwith DCD-MvDN30.1 or DCDMvDN30.2 or MvDN30. FIGS. 10A-10D human gastriccarcinoma cell lines; FIGS. 10E-10F non small cell lung carcinoma celllines. Cells were plated in 96 well costar (1000/well) in 5% FCS medium.After 24 hrs cells were treated with increasing concentrations of thedifferent molecules for further 72 hrs. Number of cells was evaluated byCell titer-glo (Promega). The plots represent the percentage of alivecells respect to untreated control. Each point is the mean of triplicatevalues. The new molecules efficiently inhibit cell growth ofMet-addicted cells.

FIG. 11: Anchorage-independent growth of cells treated with DCD-MvDN30.1or DCD-MvDN30.2 or MvDN30. A549 cells were plated in semi-solid medium(5% agarose) with or without HGF (50 ng/ml) in the presence of 1.5 mM ofDCD-MvDN30.1, or DCD-MvDN30.2 or MvDN30. After 21 days colonies werestained with tetrazolium salt. Colonies were quantified by countingpixel in each well area with MetaMorphOffline Software. Each point isthe mean of triplicate values. The new molecules efficiently inhibitHGF-dependent anchorage-independent cell growth.

FIG. 12: Pharmakokinetic profile in vivo of DCD-MvDN30.1, DCD-MvDN30.2and MvDN30. Immunodeficient mice were injected intraperitoneous with asingle dose (100 μg) of DCD-MvDN30.1, or DCD-MvDN30.2 or MvDN30.Peripheral blood was collected at different time points. Serumconcentrations of the therapeutic molecules were measured by ELISA.Graph represents the amount of circulating molecules in function oftime. Samples are in triplicate, bars represent standard deviations.

FIGS. 13A-13B: Nucleotide and amino acid sequences of a first embodimentof a first polypeptide of an antibody fragment according to the presentdisclosure. The sequences correspond to the polypeptide derived from thelight chain, VL-CL-CL. The CDR regions are underlined both in thenucleotide and amino acid sequences.

FIGS. 14A-14B: Nucleotide and amino acid sequences of a first embodimentof a second polypeptide of an antibody fragment according to the presentdisclosure. The sequences correspond to the polypeptide derived from theheavy chain, VH-CH1-CH1-TAGs. The CDR regions are underlined both in thenucleotide and amino acid sequences. Strep and Histidine TAGs in capitalitalic letters.

FIGS. 15A-15B: Nucleotide and amino acid sequences of a secondembodiment of a first polypeptide of an antibody fragment according tothe present disclosure. The sequences correspond to the polypeptidederived from the light chain, VL-CL-CH1-TAGs. The CDR regions areunderlined both in the nucleotide and amino acid sequences. Strep andHistidine TAGs in capital italic letters.

FIGS. 16A-16B: Nucleotide and amino acid sequences of a secondembodiment of a second polypeptide of an antibody fragment according tothe present disclosure. The sequences correspond to the polypeptidederived from the heavy chain, VH-CH1-CL. The CDR regions are underlinedboth in the nucleotide and amino acid sequences.

FIGS. 17A-17B: Nucleotide and amino acid sequences of DN30 light chainvariable domain. The CDR regions are underlined both in the nucleotideand amino acid sequences.

FIGS. 18A-18B: Nucleotide and amino acid sequences of DN30 heavy chainvariable domain. The CDR regions are underlined both in the nucleotideand amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail, by way of non limitingexample, with reference to antibody fragments able to specifically bindhepatocyte growth factor receptor.

It is clear that the scope of this description is in no way limited tosuch target antigen, since the antibody fragments described herein canbe characterized by specifically binding other target antigens.

In the following description, numerous specific details are given toprovide a thorough understanding of embodiments. The embodiments can bepracticed without one or more of the specific details, or with othermethods, components, materials, etc. In other instances, well-knownstructures, materials, or operations are not shown or described indetail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment. Thus, the appearances of the phrases “in oneembodiment” or “in an embodiment” in various places throughout thisspecification are not necessarily all referring to the same embodiment.Furthermore, the particular features, structures, or characteristics maybe combined in any suitable manner in one or more embodiments.

The headings provided herein are for convenience only and do notinterpret the scope or meaning of the embodiments.

Antibodies are complex tetramers in which both the heavy and the lightchains are composed by multiple Ig domains, each one foldingindependently. At the very beginning of the antibody era it has beenshown that, through enzymatic treatment, an antibody can originatefragments that maintain the original structure and the antigen-bindingproperties.

Subsequently, by applying protein engineering techniques, a plethora ofdifferent engineered antibody fragments have been generated. Accordingto molecular design, each new antibody fragment is characterized byparticular features (i.e. increased avidity, multivalency,multispecificity, ADCC-deficient, chimerized, etc.).

However, none of the previous studies have addressed the issue of renalclearance to prolong half-life in vivo of antibody fragments.

To this end, the present inventors developed a recombinant antibodyfragment comprising a first polypeptide comprising a light chainvariable domain and two constant domains and a second polypeptidecomprising a heavy chain variable domain and two constant domains,wherein two chain constant domains are light chain constant domains andtwo constant domains are heavy chain CH1 constant domains, fused indifferent combinations to the variable domains.

In one embodiment, the antibody fragment herein disclosed is more stablein vivo than the Fab molecule comprising said light and heavy chainvariable domains.

In one embodiment, the antibody fragment has prolonged half-life in vivowhen administered to a human patient than the Fab molecule comprisingsaid light and heavy chain variable domains because of a reduced renalclearance.

These antibody fragments were named ‘Dual Constant Domain Fabs’(DCD-Fabs).

In a preferred embodiment, the present disclosure concerns an antibodyfragment comprising a first polypeptide comprising a light chainvariable domain and two constant domains and a second polypeptidecomprising a heavy chain variable domain and two constant domains,wherein two constant domains are human light chain constant domains andtwo constant domains are human heavy chain CH1 constant domains, fusedin different combinations to the variable domains, wherein the antibodyfragment is more stable in vivo than the Fab molecule comprising saidlight and heavy chain variable domains, and wherein the antibodyfragment specifically binds the hepatocyte growth factor receptor(HGFR/Met).

In one embodiment, the light chain variable domain is fused at itsC-terminus to one light chain constant domain, that is fused at itsC-terminus to one light chain constant domain.

In another embodiment, the light chain variable domain is fused at itsC-terminus to a light chain constant domain, that is fused at itsC-terminus to one heavy chain CH1 constant domain.

In one embodiment, the heavy chain variable domain is fused at itsC-terminus to one heavy chain CH1 constant domain, that is fused at itsC-terminus to one heavy chain CH1 constant domain.

In another embodiment, the heavy chain variable domain is fused at itsC-terminus to a heavy chain CH1 constant domain, that is fused at itsC-terminus to a light chain constant domain.

In one embodiment, the constant domains contained in the first andsecond polypeptide—when coupled together in the antibody fragment—areable to generate disulfide bridges.

In a further preferred embodiment, the present disclosure concernsantibody fragments as defined above wherein the antigen specificity isprovided by employing as light and heavy chain variable domains the DN30light and heavy chain variable domains or humanized light and heavychain variable domains comprising the complementarity determiningregions (CDRs) from DN30 monoclonal antibody. DN30 monoclonal antibodywas disclosed in the international patent application WO-A-2007/090807.

An antibody fragment of the invention is generally a therapeuticantibody. For example, an antibody of the invention may be anantagonistic antibody, a blocking antibody or a neutralizing antibody.

In one aspect, the invention provides methods of treating or delayingprogression of a disease administering to a subject having the diseasean effective amount of an antibody fragment of the invention, effectivein treating or delaying progression of the disease.

In one embodiment, the disease is a tumor or tumor metastasis.

In another embodiment, the disease is associated with dysregulation ofhepatocyte growth factor-receptor signalling and/or activation.

An antibody fragment of the invention is suitable for treating orpreventing pathological conditions associated with abnormalities withinthe HGF/HGFR signalling pathway.

In one embodiment, an antibody of the invention is a HGFR antagonist.

In one embodiment, the antibody fragment comprises antigen bindingsequences from a non-human donor grafted to a heterologous non-human,human or humanized sequence (e.g. framework and/or constant domainsequences). In one embodiment, the non-human donor is a mouse.

In one embodiment, the antigen binding sequences comprise all the CDRsand/or variable domain sequences of an anti-HGFR murine antibody.

In one preferred embodiment, the murine light chain variable domain isfused at its C-terminus to one human kappa light chain constant domain,that is fused at its C-terminus to one human kappa light chain constantdomain. In another embodiment, the murine light chain variable domain isfused at its C-terminus to a human kappa light chain constant domain,that is fused at its C-terminus to one human IgGl heavy chain CH1constant domain. In one embodiment, the murine heavy chain variabledomain is fused at its C-terminus to one human IgGl heavy chain CH1constant domain, that is fused at its C-terminus to one human IgGl heavychain CH1 constant domain. In another embodiment, the murine heavy chainvariable domain is fused at its C-terminus to a human IgGl heavy chainCH1 constant domain, that is fused at its C-terminus to a human kappalight chain constant domain.

In one preferred embodiment, an antibody fragment of the inventioncomprises a first polypeptide comprising a light chain variable domaincomprising the CDR sequences of an anti-HGFR murine antibody, morepreferably the CDRs of DN30, and two constant domains, wherein the twoconstant domains are: two light chain constant domains or one lightchain constant domain and one heavy chain CH1 constant domain. In oneembodiment the two constant domains are human constant domains.

In one embodiment, an antibody fragment of the invention comprises asecond polypeptide comprising a heavy chain variable domain comprisingthe CDR sequences of an anti-HGFR murine antibody, more preferably theCDRs of DN30, and two constant domains, wherein the two constant domainsare: two heavy chain CH1 constant domains or one heavy chain CH1constant domain and one light chain constant domain. In one embodimentthe two constant domains are human constant domains.

The invention provides, in a most preferred embodiment, a humanizedantibody fragment that binds human HGFR, wherein the antibody iseffective to inhibit HGF/HGFR activity in vivo, the antibody comprisingi) in the heavy chain variable domain (VH) the three CDRs sequence ofthe heavy chain variable domain of the DN30 monoclonal antibody (SEQ IDNos.:19,21,23) and substantially a human consensus sequence e.g.substantially the human consensus framework (FR) residues of human heavychain subgroup and ii) in the light chain variable domain (VL) the threeCDRs sequence of the light chain variable domain of the DN30 monoclonalantibody (SEQ ID Nos.:25,27,29) and substantially the human consensusframework (FR) residues of human light chain K subgroup I (VKI).

In one embodiment, an antibody fragment of the invention comprises afirst polypeptide comprising as the light chain variable domain thelight chain variable domain sequence set forth in SEQ ID NO: 12 (DN30light chain variable domain) and a second polypeptide comprising asheavy chain variable domain the heavy chain variable domain sequence setforth in SEQ ID NO: 4 (DN30 heavy chain variable domain).

In one aspect, the invention provides for use of an antibody fragment ofthe invention (e.g. a HGFR antagonist antibody fragment of theinvention) in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder.

In one aspect, the invention provides a method of treating apathological condition associated with dysregulation of HGFR activationin a subject, said method comprising administering to the subject aneffective amount of a HGFR antagonist antibody fragment of theinvention, whereby said condition is treated.

In one aspect, the invention provides a method of inhibiting the growthof a cell that expresses HGFR, said method comprising contacting saidcell with a HGFR antagonist antibody fragment of the invention therebycausing an inhibition of growth of said cell.

In one aspect, the invention provides a method of therapeuticallytreating a mammal having a cancerous tumor comprising a cell thatexpresses HGFR, said method comprising administering to said mammal aneffective amount of a HGFR antagonist antibody fragment of theinvention, thereby effectively treating said mammal.

In one aspect, the invention provides a method for treating orpreventing a cell proliferative disorder associated with increasedexpression or activity of HGFR, said method comprising administering toa subject in need of such treatment an effective amount of a HGFRantagonist antibody fragment of the invention, thereby effectivelytreating or preventing said cell proliferative disorder.

In one aspect, the invention provides a method of therapeuticallytreating a tumor in a mammal, wherein the growth of said tumor is atleast in part dependent upon a growth potentiating effect of HGFR, saidmethod comprising contacting a tumor cell with an effective amount of aHGFR antagonist antibody fragment of the invention, thereby effectivelytreating said tumor. The tumor cell can be one selected from breast,colorectal, lung, colon, pancreatic, prostate, ovarian, cervical,central nervous system, renal, hepatocellular, bladder, gastric, headand neck tumor cell, papillary carcinoma (e.g. the thyroid gland),melanoma, lymphoma, myeloma, glioma/glioblastoma (e.g. anaplasticastrocytoma, glioblastoma multiforme, anaplastic oligodendroglioma,anaplastic oligodendroastrocytoma), leukemia cell. In one embodiment, acell that is targeted in a method of the invention is ahyperproliferative and/or hyperplastic cell. In one embodiment, a cellthat is targeted in a method of the invention is a dysplastic cell. Inyet another embodiment, a cell that is targeted in a method of theinvention is a metastatic cell. In a further embodiment, a cell that istargeted in a method of the invention is a HGFR expressing cellbelonging to the microenvironment sustaining the tumor and/or themetastasis.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted tumor cell and/or tissue is exposed to radiationtreatment or a chemotherapeutic agent. In another embodiment, a targetedtumor cell and/or tissue is treated, in addition to the antagonistantibody fragment of the invention, with HGF inhibitors (i.e. anti-HGFantibodies) or other anti-HGFR compounds (i.e. small molecule kinaseinhibitors). In a further embodiment, a targeted tumor cell and/ortissue is treated, in addition to the antagonist antibody fragment ofthe invention, with molecules specifically hitting other targetsrelevant in the maintenance of the transformed phenotype (i.e. anti-EGFRmolecules).

Activation of HGFR is an important biological process; its deregulationleads to numerous pathological conditions. Accordingly, in oneembodiment of methods of the invention, a cell that is targeted (e.g. acancer cell) is one in which activation of HGFR is enhanced as comparedto a normal cell of the same tissue origin. In one embodiment, a methodof the invention causes the death or cell growth arrest of a targetedcell. For example, contact with an antagonist antibody fragment of theinvention may result in a cell's inability to signal through the HGFRpathway, which results in cell death or cell growth arrest.

The invention also pertains to immunoconjugates, or antibody-drugconjugates (ADC), comprising an antibody fragment conjugated to acytotoxic agent such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e. a radioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells growthin the treatment of cancer, allows targeted delivery of the drug moietyto tumors, and intracellular accumulation therein, where systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated.

Therapeutic formulations comprising an antibody fragment of theinvention are prepared for storage by mixing the antibody fragmenthaving the desired degree of purity with physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions,lyophilized or other dried formulations. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers; antioxidants;preservatives; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids; monosaccharides, disaccharides, and other carbohydrates;chelating agents; sugars; salt-forming counter-ions; metal complexesand/or non-ionic surfactants.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared bymeans of techniques disclosed i.a. in Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody fragment of the invention,which matrices are in the form of shaped articles, e.g. films, ormicrocapsule.

An antibody fragment of the present invention may be used in in vitro,ex vivo and in vivo therapeutic methods. The invention provides variousmethods based on using antibody fragments having superior propertiescompared to conventional monovalent antibodies.

The present invention provides antibody fragments, which can be used fora variety of purposes, for example as therapeutics, prophylactics anddiagnostics.

Antibody fragments of the invention can be used either alone or incombination with other compositions in a therapy. For instance, anantibody fragment of the invention may be co-administered with anotherantibody, chemotherapeutic agent(s) (including cocktails ofchemotherapeutic agents), other cytotoxic agent(s), anti-angiogenicagent(s), cytokines, and/or growth inhibitory agent(s). Such combinedtherapies noted above include combined administration (where the two ormore agents are included in the same or separate formulations), andseparate administration, in which case, administration of the antibodyof the invention can occur prior to, and/or following, administration ofthe adjunct therapy or therapies.

The antibody fragment of the invention (and adjunct therapeutic agent)is/are administered by any suitable means, including parenteral,subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. The antibodyfragment is suitably administered by pulse infusion, particularly withdeclining doses of the antibody. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic. Theantibody fragment of the invention can be also delivered by genetransfer by mean of viral vectors (i.e. lentiviral vectors),administered locally or systemically.

The antibody fragment of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount ofantibodies of the invention present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

For the prevention or treatment of disease, the appropriate dosage of anantibody fragment of the invention (when used alone or in combinationwith other agents such as chemotherapeutic agents) will depend on thetype of disease to be treated, the type of antibody, the severity andcourse of the disease, whether the antibody fragment is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody fragment is suitably administered tothe patient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 mg/kg to 15 mg/kg of antibodyis an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. One typical daily dosage might range from about 1mg/kg to 100 mg/kg or more, depending on the factors mentioned above.For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. One exemplary dosage of the antibody fragmentwould be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, oneor more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, e. g. every week or every three weeks(e. g. such that the patient receives from about two to about twenty, e.g. about six doses of the antibody). An initial higher loading dose,followed by one or more lower doses may be administered. An exemplarydosing regimen comprises administering an initial loading dose of about4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of theantibody. However, other dosage regimens may be useful. The progress ofthis therapy is easily monitored by conventional techniques and assays.

Results

Generation of the Chimeric DN30 Fab and Characterization of itsBiochemical and Biological Properties.

Like other monoclonal antibodies with therapeutic potential, DN30 hasbeen raised in mice. Thus, its direct employment in humans fortherapeutic purpose is not applicable, as the murine molecule would berecognized by human anti-murine antibodies (HAMA) that leads toimmuno-mediated clearance of the antibody activity. Substitution of themurine constant regions of the antibody with sequences derived fromhuman immunoglobulins (antibody chimerization) is sufficient to stronglyreduce the HAMA response. Chimerized mAbs and Fabs are currently used inthe clinic. Through classical molecular biology techniques, the presentinventors have substituted the constant domains of the DN30 Fab heavyand light chains with constant domains derived from humanimmunoglobulins: the light chain constant domain has been substitutedwith the human kappa type domain, the one more represented in thenatural human antibodies, while the heavy chain CH1 constant domain hasbeen substituted with the homologous domain derived from the human IgGl.This combination is effective: the chimerized DN30 Fab (MvDN30) bindsMet with high affinity, induce Met shedding and inhibits proliferationof Met-addicted cells, overlapping the properties of the correspondingmurine molecule (FIG. 1 and FIG. 2).

Molecular Design of the Dual Constant Domain Fab.

Using the MvDN30 sequence as a template, the present inventorsduplicated the constant domains in each light and heavy chain (DualConstant Domain-Fab). The new engineered molecule has a predictedmolecular weight of 75 kD. The present inventors generated two differentDCD-Fabs. In the first molecule the human constant domains wereduplicated in tandem, thus generating a VH-CH1-CH1 chimeric heavy chainand a VL-CL-CL chimeric light chain. In the second molecule the terminaldomain were swapped reciprocally, thus generating a VH-CH1-CL chimericheavy chain and a VL-CL-CH1 chimeric light chain (FIG. 3). Thecorresponding dimeric recombinant molecules were named DCD-MvDN30.1 andDCD-MvDN30.2. cDNAs encoding for these new molecules were cloned into anexpression plasmid and then expressed into eukaryotic cells. Proteinwere purified from cell culture supernatant thank to the StrepTAG thatwas inserted at the C-terminus of the sequence. FIG. 4 shows theSDS-Page separation under reducing condition of the purified recombinantmolecules having the correct molecular weight size.

DCD-MvDN30.1 and DCD-MvDN30.2 Bind to Met with High Affinity.

Purified DCD-MvDN30.1 and DCD-MvDN30.2 were characterized for theirability to bind the Met receptor. To this end, the present inventorsperformed ELISA assays using Met ectodomain in solid phase and MvDN30,DCD-MvDN30.1 and DCD-MvDN30.2 in liquid phase. Binding was revealedusing anti-strepTAG antibodies (FIG. 5). This analysis showed that thethree DN30-derived monovalent molecules bind to Met with a similaraffinity (MvDN30, K_(d)=0.141±0.03 nM; DCD-MvDN30.1, K_(d)=0.133±0.02nM; DCD-MvDN30.2, K_(d)=0.130±0.03 nM).

DCD-MvDN30.1 and DCD-MvDN30.2 do not Induce Met Phosphorylation.

The present inventors tested whether the new DN30-derived moleculescould display a Met agonistic activity in Met phosphorylation assay.This was analysed using A459 human lung carcinoma cells, which representa standard system for determining Met activation in response to acuteligand stimulation. In fact, A549 cells express physiological levels ofMet, inactive in basal conditions, but prone to be activated by HGF or aligand-mimetic molecule (4, 10). Cells were stimulated for 15 minuteswith increasing amounts of MvDN30, DCD-MvDN30.1 and DCD-MvDN30.2. Cellswere also stimulated with HGF and DN-30 mAb as positive controls. Metactivation was determined by immunoblotting with anti-phosphoMetantibodies. As shown in FIG. 6, the new molecules did not show anysignificant agonistic activity. DCD-MvDN30.1 was indistinguishable fromMvDN30, being devoid of any agonistic activity. DCD-MvDN30.2 retained aminimal residual agonist activity, which was in any case negligiblecompared with the DN30 mAb or HGF. The present inventors also checkedthe activation of molecules acting as downstream effectors of Met. Whilestimulation with HGF induced the activation of both Extracellularsignal-Regulated Kinases 1 and 2 (ERK-1 and ERK-2) and AKT/ProteinKinase B (AKT), DCD-MvDN30.1 and DCD-MvDN30.2, as MvDN30, did not affectthe phosphorylation status of these signal transducers (FIG. 7).

DCD-MvDN30.1 and DCD-MvDN30.2 Induce Met Shedding.

The present inventors also investigated whether the new moleculesderived from MvDN30 maintain the ability to promote receptor sheddingand downregulation. A549 cells were incubated with DCD-MvDN30.1,DCD-MvDN30.2 and MvDN30. After 48 hours, the presence of Met ectodomainin the conditioned medium was analyzed by immunoblotting using amonoclonal antibody directed against the extracellular portion of Met.Total cellular levels of Met were also determined on cell lysates usingthe same antibody. This analysis revealed that both DCD-MvDN30.1 andDCD-MvDN30.2 efficiently induced Met shedding and promoted Metdown-regulation, resulting in release of soluble Met ectodomain in theextracellular space and decreased Met levels in the cell (FIG. 8).Therefore, the new MvDN30 derived molecules, like MvDN30, achievedcomplete disassociation between the antagonistic and agonisticproperties of the parental DN-30 mAb.

DCD-MvDN30.1 and DCD-MvDN30.2 Inhibit HGF-Induced Met Phosphorylationand Down-Stream Signaling.

The present inventors investigated if DCD-MvDN30.1 and DCD-MvDN30.2could inhibit HGF-induced Met phosphorylation and down-stream signaling.A549 cells were incubated with DCD-MvDN30.1, DCD-MvDN30.2 and MvDN30 for24 hrs and then stimulated for 15 minutes with HGF. Met activation wasdetermined by immunoblotting with anti-phosphoMet antibodies. As shownin FIG. 9, the two engineered molecules, as MvDN30, efficientlydown-regulated Met receptor and strongly impaired the level of itsphosphorylation. This resulted in an inhibition of AKT and ERK-1,2activation (FIG. 9).

DCD-MvDN30.1 and DCD-MvDN30.2 Inhibit MET-Addicted Anchorage-DependentCell Growth.

Anchorage-dependent growth can be impaired by a Met-inhibitor only inthe cells that rely on Met-signalling for proliferation/survival, the socalled MET-addicted cells. The present inventors analysed the completepanel of MET-addicted tumor cells (GTL-16, SNU-5, Hs746T, MKN-45—humangastric carcinoma cells—and H1993, EBC-1—human lung carcinoma cells).Exponentially growing cells were incubated with increasingconcentrations of DCD-MvDN30.1 and DCD-MvDN30.2. MvDN30 was included inthe assay as positive control. After 72 hours cell growth was determinedusing a luminescence-based ATP assay. Both the MvDN30-derived moleculesinhibited all the MET-addicted cell growth in a dose-dependent fashion(FIG. 10). Inhibitory properties of the two DCD molecules werecomparable to the ones of MvDN30 in all cells tested.

DCD-MvDN30.1 and DCD-MvDN30.2 Inhibit Anchorage-Independent Cell Growth.

The present inventors tested the ability of DCD-MvDN30.1 andDCD-MvDN30.2 to inhibit anchorage independent growth of A549 cells.Cells were seeded in semi-solid medium incubated or not with HGF andtreated with a single dose of DCD-MvDN30.1, DCD-MvDN30.2 and MvDN30.After two weeks, cell colonies were stained and quantified. In thisassay as well, DCD-MvDN30.1 and DCD-MvDN30.2 reduced HGF-dependentcolony formation in a fashion similar to that of MvDN30 (FIG. 11).

DCD-MvDN30.1 and DCD-MvDN30.2 Show Improved Pharmacokinetic Profile InVivo Compared to MvDN30.

The present inventors studied the pharmacokinetic properties ofDCD-MvDN30.1 and DCD-MvDN30.2, in comparison with MvDN30. A single doseof the above mentioned molecules were delivered by intraperitonealinjection to immunodeficient mice. Peripheral blood from the treatedmice was collected at different time points after the delivery. Thecirculating concentrations of the studied molecules were determined byELISA performed on the serum samples. DCD-MvDN30.1 and DCD-MvDN30.2reached higher circulating levels compared to MvDN30. Moreover both themolecules showed increased half-life and are longer lasting in thecirculation, being biological available for a longer time. DCD-MvDN30.1and DCD-MvDN30.2 clearance is strongly improved compared to MvDN30(clearance reduction compared to MvDN30: 9.6 and 13.7 fold respectivelyfor DCD-MvDN30.1 and DCD-MvDN30.2) (FIG. 12 and Table 1).

TABLE 1 Pharmacokinetic parameters of the different DN30-derivedmolecules CL Vss Cmax Tmax AUCtot kel t½ (ml/h) (ml) (ng/ml) (h)(ng/ml)h (1/h) MVDN30 8.41 4.36 11.96 7595 0.5 22933 0.082 DCD- 10.530.45 5.77 24130 4 220188 0.066 MvDN30.1 DCD- 10.27 0.32 4.36 24952 4314667 0.068 MvDN30.2 t½: half-life; CL: clereance; Vss: Volume ofdistribution; Cmax: maximal molecule concentration; Tmax: time to reachCmax; AUCtot: area under the Curve; Kel: constant of elimination.Material and MethodsCell Culture

EBC-1 human lung carcinoma cell and MKN-45 gastric carcinoma cell linewere obtained from the Japanese Collection of Research Bioresources(Osaka, Japan). GTL-16 human gastric carcinoma cells were derived fromMKN-45 cells as described (11). All other cell lines were obtained fromthe ATCC-LGC Standards partnership (Sesto San Giovanni, Italy). All celllines were maintained in RPMI except Hs746T—in DMEM—and SNU-5—in IMDM.Cell media were supplemented with 10% (20% for SNU-5) Fetal Bovine Serumand 2 mM glutamine (Media, serum and glutamine were from Sigma LifeScience, St. Louis, Mo.).

Protein Engineering

DCD-MvDN30.1 and DCD-MvDN30.2 are comprised of a heavy chain and a lightchain.

The DCD-MvDN30.1 heavy chain (SEQ ID NO.:1 and 2) corresponds to the VHdomain of wild-type DN30 Fab (2; SEQ ID No.: 3 and 4) fused to the CH1domain of human immunoglobulin G1 repeated in tandem (SEQ ID NO.: 5 and6). At the C-terminus, a STREP tag (ST, SEQ ID NO.:7) and apoly-histidine tag (HT, SEQ ID NO.:8) have been added for purificationand detection purposes. The overall structure corresponds to (from theN- to the C-terminus): VH-CH1-CH1-ST-HT. The nucleotide and amino acidsequences of the heavy chain of DCD-MvDN30.1 are reported in FIGS. 14Aand B, respectively.

The DCD-MvDN30.1 light chain (SEQ ID No.:9 and 10) corresponds to the VLdomain of wild-type DN30 Fab (SEQ ID No.:11 and 12) fused to the CLdomain of human immunoglobulin kappa (SEQ ID NO.:13 and 14) repeated intandem. The overall structure corresponds to (from the N- to theC-terminus): VL-CL-CL. The nucleotide and amino acid sequences of thelight chain of DCD-MvDN30.1 are reported in FIGS. 13A and B,respectively.

The DCD-MvDN30.2 heavy chain (SEQ ID No.:15 and 16) corresponds to theVH domain of wild-type DN30 Fab (SEQ ID No.:3 and 4) fused to the CH1domain of human immunoglobulin G1 (SEQ ID NO.: 5 and 6) plus the CLregion of human immunoglobulin kappa (SEQ ID NO.:13 and 14). The overallstructure corresponds to (from the N- to the C-terminus): VH-CH1-CL. Thenucleotide and amino acid sequences of the heavy chain of DCD-MvDN30.2are reported in FIGS. 16A and B, respectively.

The DCD-MvDN30.2 light chain (SEQ ID No.:17 and 18) corresponds to theVL domain of wild-type DN30 Fab (SEQ ID No.:11 and 12) fused to the CLdomain of human immunoglobulin kappa (SEQ ID NO.:13 and 14) plus the CH1of human immunoglobulin G1 (SEQ ID NO.: 5 and 6) plus the STREP tag (ST,SEQ ID NO.:7) and the poly-histidine tag (HT, SEQ ID NO.:8). The overallstructure corresponds to (from the N- to the C-terminus):VL-CL-CH1-ST-HT. The nucleotide and amino acid sequences are of thelight chain of DCD-MvDN30.2 reported in FIGS. 15A and B, respectively.

The cDNAs encoding DCD-MvDN30.1 and DCD-MvDN30.2 were synthesizedchemically by the GeneArt® service (Life Technologies, Paisley, UnitedKingdom). The nucleotide and amino acid sequences of DN30 Fab light andheavy chain variable domains are reported in FIGS. 17 and 18,respectively. The CDR regions are underlined both in the nucleotide andamino acid sequences, wherein the CDRs of the heavy chain variabledomain have the amino acid and nucleotide sequences set forth in SEQ IDNos.:19,21,23 and 20,22, respectively, and the CDRs of the light chainvariable domain have the amino acid and nucleotide sequences set forthin SEQ ID Nos.:25, 27,29 and 26,28,30 respectively.

All constructs were engineered to contain a BamHI site at the 5′ end anda NotI site at the 3′ end. The BamHI-NotI fragments were subcloned intothe pUPEX expression vector (U-Protein Express, Utrecht, TheNetherlands). Medium-scale production of DCD-MvDN30.1 and DCD-MvDN30.2was outsourced to U-Protein Express that achieved it by transienttransfections into HEK (Human Epithelial Kidney) cells. Proteins werepurified by affinity chromatography using the STREP tag and thepoly-histidine tag. Purified proteins were conserved in PBS plus 0.02%Tween-80 (Sigma-Aldrich) and stored at 4° C. Purity was determined bySDS-PAGE in both reducing and non-reducing conditions followed byCoomassie staining.

Immunoprecipitation and Western Blotting

Immunoprecipitation was performed as described (12) using the DO-24anti-Met mAb (4). Western blotting was performed using the followingantibodies: anti-human Met mAb clone DL-21 that recognizes a domainlocated in the extracellular portion of Met (4); anti-phosphotyrosinemAb clone 4G10 mAb (Millipore, Temecula, Calif.); anti-phospho-Met (Tyr1234/1235), anti-phospho-Met (Tyr 1349), anti-phospho-Akt (Ser 473),anti-Akt, anti-phospho-ERK (Thr 202/Tyr 204) and anti-ERK polyclonal Abs(Cell Signaling Technology, Beverly, Mass.).

ELISA Binding Assays

Binding of MvDN30, DCD-MvDN30.1 and DCD-MvDN30.2 was determined by ELISAusing a Met-Fc chimera in solid phase (R&D Systems, Minneapolis, Minn.)and increasing concentrations of FLAG-tagged recombinant antibody inliquid phase. Binding was revealed using an anti-strepTAG II antibodyconjugated with horseradish peroxidase (IBA, Olivette, Mo.). Data wereanalyzed and fit using Prism software (Graph Pad Software, San Diego,Calif.). Met-Fc chimera is a fusion protein wherein the Fc domainderived from a human IgG is fused in frame with the Met extracellularportion.

Met Activation Analysis

Subconfluent A549 human lung carcinoma cells were incubated inserum-free medium for 48 hours and then stimulated for 10 minutes withthe indicated concentrations of recombinant HGF (R&D Systems) orpurified DN-30 mAb, MvDN30, DCD-MvDN30.1 and DCD-MvDN30.2 as described(13). Following stimulation, cells were immediately lysed and processedas described (12). Cell extracts were immunoprecipitated with anti-Metantibodies (D0-24), resolved by SDS-PAGE and analyzed by Westernblotting using anti-phosphotyrosine antibodies (Millipore). The sameblots were re-probed with anti-Met antibodies (DL-21) to normalize theamount of Met immunoprecipitated.

For the inhibition of HGF-induced Met phosphorylation A549 cells weretreated for 24 hrs in serum free medium with MvDN30, DCD-MvDN30.1 andDCD-MvDN30.2 and than stimulated with HGF as described above. Cellmonolayers were lysated with Laemmli buffer and equal amounts of totalproteins, separated into acrylammide gel by SDS-PAGE and analyzed byimmunoblotting with anti-phospho-Met (Tyr 1234/1235) antibodies.

Analysis of Met Shedding

Subconfluent A549 monolayers were washed twice with PBS and thenincubated in serum-free medium with the indicated concentrations ofDN-30 FAb or mAb. After hours, conditioned medium was collected andcells were lysed with Laemmli Buffer. Met protein levels were determinedin 50 μg of total cell lysates and in 50 μl of cell culture supernatantby Western blotting using the anti-Met DL-21 mAb.

In Vitro Biological Assays

For cell growth analysis, cells were seeded in 96 well-dishes (1,000cells/well) in medium containing 10% FBS. After 24 hours, the medium wasreplaced with fresh one containing the DN30-derived molecules plus 5%FCS antibodies at the indicated concentrations. Cell number wasevaluated after 72 hrs using the CellTiter-Glo luminescent cellviability assay (Promega Corporation, Madison, Wis.) according tomanufacturer's instructions. Chemo-luminescence was detected with aMultilabel Reader PerkinElmer 2030 apparatus (PerkinElmer Life andAnalytical Sciences, Turku, Finland).

For anchorage-independent growth assays, cells were seeded in 48well-dishes (500 cells/well) in medium containing 2% FBS and 0.5%SeaPlaque agarose (BMA, Rockland, Me.). Antibodies (1.5 μM) and HGF (50ng/ml) were added in the culture medium every 3 days. After 21 days ofculture, colonies were stained by tetrazolium salts (Sigma Life Science)and scored by MetaMorphOffline Software (Molecular Device LLC,Sunnyvale, Calif.).

Pharmakokinetic Analysis

Adult immunodeficient NOD-SCID mice (body weight between 18 and 22 gr,on average 20 gr) were injected IP with 100 μg of DCD-MvDN30.1 orDCD-MvDN30.2 or MvDN30. Peripheral blood was collected at different time(for MvDN30: 10, 20 and 30 min, 1, 2, 4, 6, 8, 10, 16, 24, 48 hours; forDCD-MvDN30.1 and DCD-MvDN30.2: 30 min, 1, 2, 4, 6, 8, 10, 16, 24, 48,72, 96, 144 hrs after the delivery). Therapeutic molecule concentrationswere evaluated by ELISA as described above in binding assay section,interpolating the absorbance values of the samples on the linear part ofa standard curve obtained by serial dilutions of the different purifiedmolecules. Each time point was the average value of a least 3 mice.

REFERENCES

-   1) Martens, T., et al. A novel one-armed anti-c-Met antibody    inhibits glioblastoma growth in vivo. Clin Cancer Res. (2006); 12:    6144-52.-   2) Jin, H., et al. MetMAb, the one-armed 5D5 anti-c-Met antibody,    inhibits orthotopic pancreatic tumor growth and improves survival.    Cancer Res. (2008); 68: 4360-8.-   3) www.clinicaltrial.gov; Identifier: NCT01456325. “A study of    Onartuzumab (MetMab) in combination with Tarceva (Erlotinib) in    patients with Met diagnostic-positive Non-Small Cell Lung cancer who    have received chemotherapy for advanced or metastatic disease    (MetLung)”.

4) Prat, M., et al. Agonistic monoclonal antibodies against the Metreceptor dissect the biological responses to HGF. J Cell Sci. (1998);111: 237-47.

-   5) Petrelli, A., et al. Ab-induced ectodomain shedding mediates    hepatocyte growth factor receptor down-regulation and hampers    biological activity. Proc Natl Acad Sci USA. (2006); 103: 5090-5.-   6) Foveau, B., et al. Down-regulation of the met receptor tyrosine    kinase by presenilin-dependent regulated intramembrane proteolysis.    Mol Biol Cell. (2009); 20: 2495-507.-   7) Schelter, F, et al. A disintegrin and metalloproteinase-10    (ADAM-10) mediates DN30 antibody-induced shedding of the met surface    receptor. J Biol Chem. (2010); 285: 26335-40.-   8) Pacchiana G, et al. Monovalency unleashes the full therapeutic    potential of the DN-30 anti-Met antibody. J Biol Chem. (2010); 285:    36149-57.-   9) Reichert J M. Antibody-based therapeutics to watch in 2011. MAbs.    (2011); 3: 76-99.-   10) Michieli P, et al. An HGF-MSP chimera disassociates the trophic    properties of scatter factors from their pro-invasive activity. Nat    Biotechnol (2002); 20:488-495.-   11) Giordano S, et al. p145, A protein with associated tyrosine    kinase activity in a human gastric carcinoma cell line. Mol Cell    Biol 1988 8:3510-3517.-   12) Longati P, et al. Tyrosines1234-1235 are critical for activation    of the tyrosine kinase encoded by the MET proto-oncogene (HGF    receptor). Oncogene 1994 9:49-57.-   13) Vigna E, et al. “Active” cancer immunotherapy by anti-Met    antibody gene transfer. Cancer Res 2008 68:9176-9183

The invention claimed is:
 1. A monovalent antibody fragment comprisinga) a first polypeptide comprising one light chain variable domain and afirst and second human light chain constant domain, wherein a lightchain variable domain is fused to the first human light chain constantdomain that is fused to the second human light chain constant domain inthe N- to C-terminal direction, thus generating a VL-CL-CL chimericlight chain, and b) a second polypeptide comprising one heavy chainvariable domain and a first and a second human heavy chain CH1 constantdomain, wherein a heavy chain variable domain is fused to the firsthuman heavy chain CH1 constant domain that is fused to the second humanheavy chain CH1 constant domain, in the N- to C-terminal direction, thusgenerating a VH-CH1-CH1 chimeric heavy chain, wherein the monovalentantibody fragment specifically binds to hepatocyte growth factorreceptor (HGFR).
 2. The monovalent antibody fragment according to claim1, wherein both the first and second light chain constant domains arehuman kappa light chain constant domains.
 3. The monovalent antibodyfragment according to claim 1, wherein both the first and second heavychain CH1 constant domains are human gamma heavy chain CH1 constantdomains.
 4. The monovalent antibody fragment according to claim 1,wherein both the first and second heavy chain CH1 constant domains arefrom a human IgG1.
 5. A pharmaceutical composition comprising themonovalent antibody fragment according to claim 1, and apharmaceutically acceptable carrier.
 6. The monovalent antibody fragmentaccording to claim 1, wherein the light chain variable domain comprisesthe complementarity determining regions (CDRs) of SEQ ID NO.: 25, 27,and 29, and the heavy chain variable domain comprises thecomplementarity determining regions (CDRs) of SEQ ID NO.: 19, 21, and23.
 7. The monovalent antibody fragment of claim 1, wherein the firstpolypeptide consists of one light chain variable domain and a first andsecond human light chain constant domain, wherein the light chainvariable domain is fused to the first human light chain constant domainthat is fused to the second human light chain constant domain in the N-to C-terminal direction, thus generating a VL-CL-CL chimeric lightchain, and wherein the second polypeptide consists of one heavy chainvariable domain and a first and second human heavy chain CH1 constantdomain, wherein the heavy chain variable domain is fused to the firsthuman heavy chain CH1 constant domain that is fused to the second humanheavy chain CH1 constant domain in the N- to C-terminal direction, thusgenerating a VH-CH1-CH1 chimeric heavy chain.
 8. The monovalent antibodyfragment of claim 1, wherein the first polypeptide comprises SEQ ID NO:10, and the second polypeptide comprises SEQ ID NO:
 2. 9. A monovalentantibody fragment comprising a) a first polypeptide comprising one lightchain variable domain, one human light chain constant domain and onehuman heavy CH1 constant domain, wherein the light chain variable domainis fused to the human light chain constant domain, and the human lightchain constant domain is fused to the human heavy chain CH1 constantdomain in the N- to C-terminal direction thus generating a VL-CL-CH1chimeric light chain, and b) a second polypeptide comprising one heavychain variable domain, one human heavy chain CHI constant domain and onehuman light chain constant domain, wherein the heavy chain variabledomain is fused to the human heavy chain CH1 constant domain, and thehuman heavy chain CH1 constant domain is fused to the human light chainconstant domain in the N- to C-terminal direction, thus generating aVH-CH1-CL chimeric heavy chain, wherein the monovalent antibody fragmentspecifically binds to hepatocyte growth factor receptor (HGFR).
 10. Themonovalent antibody fragment according to claim 9, wherein the lightchain variable domain comprises the complementarity determining regions(CDRs) of SEQ ID NO.: 25, 27, and 29, and the heavy chain variabledomain comprises the complementarity determining regions (CDRs) of SEQID NO.: 19, 21, and
 23. 11. The monovalent antibody fragment of claim 9,wherein the first polypeptide consists of one light chain variabledomain, one human light chain constant domain and one human heavy CH1constant domain, wherein the light chain variable domain is fused to thehuman light chain constant domain, and the human light chain constantdomain is fused to the human heavy chain CH1 constant domain in the N-to C-terminal direction thus generating a VL-CL-CH1 chimeric lightchain, and wherein the second polypeptide comprises one heavy chainvariable domain, one human heavy chain CH1 constant domain and one humanlight chain constant domain, wherein the heavy chain variable domain isfused to the human heavy chain CH1 constant domain, and the human heavychain CH1 constant domain is fused to the human light chain constantdomain in the N- to C-terminal direction, thus generating a VH-CH1-CLchimeric heavy chain.
 12. The monovalent antibody fragment of claim 9,wherein the first polypeptide comprises SEQ ID NO: 18 and the secondpolypeptide comprises SEQ ID NO:
 16. 13. The monovalent antibodyfragment according to claim 9, wherein the light chain constant domainis a human kappa light chain constant domain.
 14. The monovalentantibody fragment according to claim 9, wherein the heavy chain CH1constant domain is a human gamma heavy chain CH1 constant domain. 15.The monovalent antibody fragment according to claim 9, wherein the heavychain CH1 constant domain is from a human IgG1.
 16. A pharmaceuticalcomposition comprising the monovalent antibody fragment according toclaim 9, and a pharmaceutically acceptable carrier.